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Astrocytes of the mouse neocortex express functional N-methyl-D-aspartate receptors ¹

Max Delbrück Center for Molecular Medicine, Cellular Neurosciences, D-13092 Berlin-Buch, Germany; and
^* Max Planck Institute for Experimental Medicine, Neurogenetics, D-37075 Göttingen, Germany

³Correspondence: Max Planck Institute of Experimental Medicine, Neurogenetics, Hermann-Rein-Str. 3, D-37075 Göttingen, Germany. E-mail: kirchhoff{at}em.mpg.de

SPECIFIC AIMS

Astrocytes are thought to be important communication partners in the central nervous system since they express a variety of transmitter receptors. To search for functional expression of the N -methyl-D -aspartate (NMDA) type of glutamate receptors in these cells, we used patch-clamp recording and calcium imaging in acutely isolated cortical brain slices of transgenic mice in which astrocytes were specifically labeled by the enhanced green fluorescent protein (EGFP).

PRINCIPAL FINDINGS

1. NMDA triggers a current response in identified protoplasmic astrocytes
To test for the presence of functional NMDA receptors in astrocytes, we generated transgenic mice in which astrocytes were labeled by EGFP under the control of the human GFAP (glial fibrillary acidic protein) promoter. EGFP-positive cells were studied in cortical areas of coronal slices from the forebrain prepared from 1- to 4-wk-old mice. Green fluorescent cells could be identified unequivocally by their morphology (small somata of about 10 µm, several processes with a bushy appearance, and contacts of end feet to blood vessels), by GFAP immunostaining, and by electrophysiological characterization of their membrane currents (Fig. 1 ). By comparing EGFP-fluorescence and phase contrast images, individual cells were approached with the patch pipette to establish the whole-cell recording mode. The membrane potential was -71.8 mV +/- 6.4 mV (n=133). NMDA triggered an inward current in the majority of EGFP-positive cells (96 of 133; Fig. 1C ). The NMDA (0.1 mM) -induced current responses ranged from 11 to 814 pA (mean 197 +/- 190 pA).

View larger version (23K): [in this window] [in a new window]   Figure 1. Morphological and electrophysiological features of EGFP-positive astrocytes in the slice. A) Confocal laser scanning microscopical image of an astrocyte from a cortical slice expressing the green fluorescent protein under the control of the GFAP promoter. Astrocytes under study were characterized by their numerous, highly branched processes. One end foot contacted a blood vessel (upper right) while other processes ended within the neuropil enwrapping synaptic regions or neuronal somata. B) Whole-cell currents of the cell shown in panel A. The cell is characterized by a symmetrical pattern of noninactivating out- and inward currents elicited by de- and hyperpolarizing voltage steps. The membrane was clamped at -80 mV. C) NMDA was applied (100 µM) to the same cell in the presence of 50 µM PDC and 50 µM CNQX.

The specific noncompetitive open channel blocker of NMDA receptors, MK-801 (1 µM), almost completely abolished the NMDA-induced current of astrocytes, to 7% as compared to a control (15 pA under MK-801 vs. 211 pA of the control, n=7). In addition, pure population of astrocytes were isolated from single-cell suspensions of brains prepared from cortices of 2-wk-old mice by fluorescence-activated cell sorting. Cellular RNA was isolated, reverse transcribed, and probed for NMDA receptor gene activity by PCR. Significant amplification signals were obtained for NR1, NR2B, and 2C.

2. The NMDA responses in astrocytes are influenced by neurones
To investigate the contribution of indirect effects triggered after activation of neuronal NMDA receptors and stimulated neuronal activity, we used pharmacological tools to block synaptic transmission (Cd²⁺, 100 µM), action potentials (tetrodotoxin, TTX, 1 to 5 µM), and glutamate uptake (PDC, L-trans-pyrrolidine-2,4-dicarboxylate, 50 µM). Cd²⁺, which is known to block presynaptic Ca²⁺ channels and thereby inhibiting Ca²⁺-dependent transmitter release, reduced the NMDA-evoked current to 23% of the control value (n=4). Similarly, blocking action potentials by TTX reduced the response to about 45% as compared to the control (n=2). In the presence of a cocktail of several blockers, i.e., TTX, PDC, Cd²⁺, and the AMPA (-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) type glutamate receptor antagonist CNQX (6-cyano-7-nitroquinoxaline-2,3-dione, 20 µM), NMDA-evoked responses were reduced to about 10 to 25% as compared to a control, but were still present. Therefore, we conclude that the majority of the NMDA-induced current is due to indirect effects via neuronal glutamate release and glial glutamate uptake, but that the remaining component points to the presence of intrinsic NMDA receptors in astrocytes.

3. Glial NMDA currents display properties of functional NMDA receptors
To study NMDA-induced changes in membrane conductance and to determine the reversal potential, we clamped the membrane potentials to a series of de- and hyperpolarizing values (-180 to +70 mV with 25 mV increment, 100 ms per voltage step). This series of voltage steps was repetitively applied every 5.5 s, which allowed us to monitor membrane conductance and determine reversal potentials at this frequency. The current voltage curve of the NMDA-induced current showed an increase in membrane conductance by 2.9 +/- 1.8 nS (n=9). The reversal potential was more positive than 25 mV. In the presence of Cd²⁺, CNQX, PDC, and TTX, the conductance increase was significantly smaller (mean 0.39 +/- 0.34 nS, n=6). The reversal potential, however, was close to zero mV (-1.3 mV, range -7.5 to +15.6 mV, n=4), as expected for responses due to the activation of NMDA receptors. The effect of the blockers (i.e., shifting the reversal potential from positive values to zero mV) indicates that the glial NMDA response is due to both, activation of glial NMDA receptors and indirect effects, most likely due to activation of glutamate transporters after neuronal glutamate release triggered by NMDA.

4. NMDA responses can be blocked by high Mg²⁺
Functional NMDA receptor channels display a voltage-dependent Mg²⁺ block at potentials more negative than -40 mV. To isolate the intrinsic astrocytic NMDA response, the experiments were performed in the presence of PDC, Cd²⁺ and CNQX. Mg²⁺ concentrations of 4 mM and higher almost abolished NMDA responses. Under these conditions, the Mg²⁺ block was irreversible and could not be washed out. However, this block could be overcome by depolarizing the membrane for 5 s to 0 mV in Mg²⁺-free solution. After such a depolarization, NMDA elicited responses with an amplitude similar to the control before application of high Mg²⁺ (n=2).

5. NMDA triggers local increases in cytosolic Ca²⁺
NMDA receptors are Ca²⁺ permeable and therefore we tested the effect of NMDA on astrocytic Ca²⁺ levels. In the first experimental series (Fig. 2A B C ), EGFP-positive cells were dialyzed with the red-shifted Ca²⁺ indicator dye calcium orange via the recording pipette solution and it was thus possible to distinguish between the emissions of the two fluorophores in our confocal system. NMDA triggered an increase in the fluorescence signal indicating an increase of intracellular [Ca²⁺] (n=3; Fig. 2A B C ). We performed the experiments in the absence or in the presence of Cd²⁺, CNQX, PDC, and TTX, but always in Mg²⁺-free bath solution while cells were clamped at -80 mV. Since calcium orange yielded only a poor signal amplitude due to its spectral properties, the recordings showed considerable noise. In a second series of experiments, therefore, we used Fluo-4, which offers a much better ratio between the Ca²⁺-bound and the free fluorophor emission (FCa²⁺-bound/FCa²⁺-free is 3 and >100 for calcium orange and Fluo-4, respectively). Since Fluo-4 and EGFP have similar fluorescence spectra, we had to record from astrocytes of nontransgenic FVB/N mice. The better properties of Fluo-4 permitted us to differentiate between responses in selected parts of processes and the soma. Whereas in the soma only small responses were detected, we recorded much larger responses in the distal part of the processes, suggesting a high density of NMDA receptors (n=8; Fig. 2D Fig. 2E ).

View larger version (40K): [in this window] [in a new window]   Figure 2. NMDA triggered an increase in [Ca2+ ]i. A) The confocal laser scanning micrograph displays the EGFP fluorescence of an astrocyte in a cortical slice obtained from a GFAP/EGFP transgenic mouse. The square indicates the sectors shown in panel B displaying the fluorescence of the Ca2+ indicator calcium orange before (upper micrograph) and during NMDA application (lower micrograph). The sector in panel B outlines the area for measuring the fluorescence change (F/Fo) in calcium orange as displayed in the upper trace in panel C ; the lower trace is the corresponding current trace that was recorded simultaneously. NMDA (100 µM) was applied as indicated by bars. D) Micrograph of Fluo-4 fluorescence: a cell with the morphological features of an astrocyte was dialyzed with Fluo-4 via the patch pipette from a nontransgenic FVB/N mouse. Note the recording pipette approaching the cell from top. Fluorescence changes (F/Fo) were analyzed in the areas marked by the squares and are displayed in the top three traces in panel E. The lower trace is the simultaneously recorded current response. NMDA (100 µM) and CNQX, TTX, Cd2+ were applied as indicated by bars.

CONCLUSIONS

For the present study, we generated a GFAP/EGFP transgenic mouse line that allowed us to unequivocally identify astrocytes. The cellular label visualized the specific astrocytic morphological features such as end feet contacting the blood vessels. The membrane currents as studied with the patch-clamp technique exhibited the passive properties as described for mature astrocytes.

We observed that a large portion of the observed NMDA current amplitude in astrocytes is blocked in the presence of drugs that interfere with neuronal activity, synaptic transmission, and glutamate uptake. We conclude that a major component of the astrocytic NMDA response is not due directly to activation of astrocytic NMDA receptors, but rather to an indirect effect involving the activation of neuronal NMDA receptors. Neuronal activity triggered in response to NMDA receptor activation could result in glial currents by the following mechanisms. 1) The increased neuronal activity leads to an increase in extracellular K⁺ and thereby triggers an inward current in astrocytes. 2) Activity-dependent release of glutamate could trigger uptake currents in glial cells. The reversal potential of the glutamate uptake current is in the positive range, which is compatible with our observation in the absence of blockers for uptake and synaptic transmission. We could isolate the intrinsic NMDA receptor response of the astrocytes by blocking the indirect effects after neuronal NMDA receptor activation and recorded a conductance increase that reversed at 0 mV. The response is blocked by MK-801, leads to an increase in cytosolic Ca²⁺, and thus shows similarities to neuronal NMDA receptors. Different from conventional neuronal or cloned NMDA receptors are reduced Mg²⁺ sensitivity and the linear current-voltage relation.

The NMDA-triggered Ca²⁺ signals in our recordings were confined to peripheral parts of processes and were not observed in the soma. Indeed, this view is supported by immunohistochemical localization of different NMDA receptor subunits at the ultrastructural level on astroglial membranes enwrapping synaptic regions. We propose that glial NMDA receptors are involved in sensing neuronal activity. Subsequently, intracellular Ca²⁺ rises trigger pathways, which induce a feedback to modulate synaptic transmission. Our Ca²⁺ recordings suggest a local role in microdomains rather than the involvement of other cell compartments such as somatic regions. Although we do not know the molecular mechanism of this feedback loop, transgenic mice with astroglial-selective NMDA receptor knockout will be valuable in our efforts to understand cortical neurotransmission.

View larger version (42K): [in this window] [in a new window]   Figure 3. Functional role of glial NMDA receptors. Distal processes of astrocytes enwrap pre- and postsynaptic structures within the cortex. Functional NMDA receptors are expressed at their membranes. Activation of these receptors leads to local rises in intracellular calcium activity. Yet-to-be identified calcium-dependent signaling pathways involving protein kinases or phosphatases could play a role as a feedback mechanism modulating synaptic transmission. Analysis of synaptic plasticity in transgenic mice with astrocyte-selective NMDA receptor knockout would increase our understanding of excitatory neurotransmission.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0439fje ; to cite this article, use FASEB J. (March 12, 2001) 10.1096/fj.00-0439fje

² Both authors contributed equally to this work.

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